Mechanisms of apoptosis in secretory and neuronal cells : role of oxidative stress and calcium overload

Abstract

Apoptosis is a widespread physiological mechanism to regulate tissue homeostasis both during development and in adult organs. However, the cell deletion program can be inappropriately activated or suppressed under pathological conditions. The present project was designed to study the role of apoptosis in the toxicity caused by oxidative stress and calcium overload.

The effects of the free radical nitric oxide (NO ) were studied in a pancreatic beta-cell line (RlNm5F). The NO-releasing compound sodium nitroprusside or interleukin-lbeta (IL-Ibeta) induced endogenous NO-production, stimulated inhibitory auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At later time-points RlNm5F cells exposed to NO underwent apoptosis. Inhibition of the nitric oxide synthase activity by NG-monomethyl-L-arginine prevented IL-1beta-induced NO generation and apoptotic cell killing. DNA-damage, caused by irradiation or free radicals, can stimulate expression of the tumor suppressor gene p53. The p53 protein is believed to produce growth arrest in G1-S which allows DNA-repair. However, such conditions have also been shown to favor the occurrence of apoptosis. We detected accumulation of the p53 protein prior to onset of apoptosis both in RINm5F cells and RAW 264.7 macrophages.

To further characterize the mechanisms involved in the deletion of pancreatic cells during oxidative stress we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). We found that DMNQ, depending on the dose, induced cell proliferation, apoptosis or necrosis. Cell proliferation was associated with induction of enzymes involved in intracellular polyaminesynthesis (i.e. ornithine decarboxylase, ODC and S-adenosyl-L-methionine decarboxylase, SAMDC). Conversely, prior to the onset of apoptosis, ODC- and SAMDC activities decreased and cells were rapidly depleted of polyamines. Interestingly, cells were protected from apoptosis when incubated with the phorbol ester TPA to induce ODC and SAMDC or by supplementing spermine.

Oxidative stress, calcium overload and NO-production have also been implicated in neuronal damage following ischemia. Thus, primary cultures of cerebellar granule cells (CGC) were used to further study the mechanisms of glutamate-induced cell killing. Part of the CGC population exposed to glutamate rapidly lost their mitochondrial membrane potential and died by necrosis. The surviving population recovered their mitochondrial membrane potential but later underwent apoptosis. Nuclear lamins were degraded prior to DNA-fragmentation, indicating an early activation of proteases in cells triggered to undergo apoptosis. Further studies suggested that protection from loss of mitochondrial membrane potential as well as inhibition of the phosphatase calcineurin play important roles in both necrotic and apoptotic neuronal death.

In conclusion, this thesis presents evidence that apoptosis is an important determinant of cell death both in pancreatic and neuronal cells exposed to oxidative stress or calcium overload. Onset of apoptosis was under different conditions associated with depletion of intracellular polyamines, accumulation of p53 or degradation of nuclear larnins. Depending on the dose and duration of exposure as well as cell sensitivity, cells died by apoptosis or necrosis in both systems used. Neuronal apoptosis was shown to be dependent on intact mitochondria supplying energy, while neurons dying by necrosis rapidly lost their mitochondrial membrane potential and were depleted of energy.